1. Field of the Invention
The present invention relates generally to an electrochemical biosensor for quantification of a specific component or analyte in liquid sample. Particularly, the present invention relates to a disposable electrochemical-based sensor for measuring the concentration of analytes in biological fluids such as plasma, serum, whole blood or the like. More particularly, the present invention relates to an electrochemical-based sensor that uses compounds as mediators for the recycling of cofactors used in the sensor.
2. Description of the Prior Art
The most common, commercially-available, amperometric biosensors are based on oxidase enzymes such as glucose oxidase, lactate oxidase, and the like. These biosensors commonly use oxygen or other mediators such as ferrocene, ferricyanate, etc., as electron acceptors that recycle the enzymes after substrate reaction. The use of dehydrogenase enzymes in biosensors, however, offer some advantages over the oxidase enzymes where the dehydrogenase enzyme-based biosensors are unaffected by oxygen during the measurement.
In recent years, analytical detection in physiological fluids, e.g. blood, urine, sweat, serum, plasma etc., is of ever increasing importance in a variety of applications including clinical laboratory testing, home testing, etc. These kinds of tests have a great impact on the diagnosis and management of various disease conditions. The development of the glucose biosensor is one of the most successful testing devices to date. Although blood glucose is clearly the most important clinical parameter to be measured in the monitoring of diabetes, it is not the only parameter of clinical interest. Other measurements of medical relevance include glycosylated hemoglobin and ketone bodies. Glycosylated hemoglobin is an indicator of long-term blood glucose levels while the level of ketone bodies is an indicator of diabetic ketoacidosis (DKA) of a patient.
The concentration of ketone bodies present in urine and blood of healthy people is very low and negligible. The presence of an excess amount of ketone bodies in blood is called ketosis. The presence of an excess amount of ketone bodies in urine is called ketonuria. Diabetes mellitus is the most common disorder associated with ketosis or ketonuria. If diabetes mellitus is untreated or inadequately treated, an excess amount of ketone bodies will accumulate in the blood, i.e ketosis, and excreted into the urine, i.e. ketonuria. This condition is called diabetic ketoacidosis (DKA). DKA is a potential life-threatening condition that occurs mostly in Type 1 diabetes, though it is also known to occur in Type 2 diabetes. Poor metabolism of high carbohydrate levels derived from insulin deficiency can result in the accumulation of ketone bodies, which is a metabolic by-product of fatty acid metabolism. Ketone bodies are produced within the mitochondria of hepatocytes and include β-hydroxybutyrate, acetoacetate, and acetone.
In diabetic ketoacidosis, β-hydroxybutyrate accounts for a major portion of the ketone bodies whereas acetone is only produced in small amounts from the decarboxylation of acetoacetate. β-hydroxybutyrate is present in blood at about two to three times the concentration of acetoacetate. Quantification of β-hydroxybutyrate levels in blood by monitoring diabetes mellitus and providing guidance for insulin therapy is helpful in avoiding DKA. Blood ketone testing, however, is more reliable for diagnosing and monitoring DKA. Conventional bedside tests for urine and blood ketone assessment, however, do not test for β-hydroxybutyrate. These tests are based on the nitroprusside reaction and react only with acetoacetate and, to a less extent, acetone. Misleading information may result from such conventional tests in the assessment of DKA.
The detection of diabetic ketoacidosis in an individual with diabetes mellitus is important and often indicates that a change in insulin dosage or other treatment is necessary. The conventional bedside tests for urine ketone assessment includes test strips provided under the trademarks Ketostix® and Keto-Diastix® (Bayer) or Chemstrip® K (Roche). Such test strips are based on non-enzyme based methods such as the nitroprusside reaction and only react with acetoacetate and to a lesser extent, acetone. These conventional test strips do not respond to β-hydroxybutyrate, which is the major concentration of ketone bodies. A disadvantage of these conventional tests is that the urine assay for ketones possesses a natural delay in detecting blood ketosis. Consequently, a urine assay for acetoacetate is insufficient to monitor the onset of ketosis in a diabetic individual. The presence of β-hydroxybutyrate in the blood indicates the onset of ketosis much earlier than the detection of acetoacetate in the urine, and is a much more accurate test for monitoring the presence of ketone bodies and, hence, the effectiveness of a particular insulin therapy.
There is an increasing need for measuring β-hydroxybutyrate, which is a dominant species of ketone bodies, in blood samples for the early and effective diagnosis of the onset of DKA. Simple, dry reagent, whole blood tests for β-hydroxybutyrate, the most clinically relevant indicator of ketoacidosis, are known in the art. A widely-used colorimetric test involves the reduction of colorless dye 2-(4-indophenyl)-3-(4-nitropheyl)-5-phenyltetrazolium chloride hydrate by β-hydroxybutyrate to produce a colored formazan compound. This common dye, however, is not stable and highly photosensitive. It also responds to ascorbate and glutathione. U.S. Pat. No. 4,254,222 (1981; Owen) and U.S. Pat. No. 4,351,899 (1982; Owen) disclose an assay for β-hydroxybutyrate where 3-hydroxybutyrate is oxidized to acetoacetate by β-hydroxybutyrate dehydrogenase (HBDH) in the presence of nicotinamide adenine dinucleatide (NAD+). The reduced NADH produced from this reaction, in turn, reacts with a tetrazolium dye to form a colored formazan compound. The degree and intensity of the color transition correlates to the concentration of β-hydroxybutyrate in the sample solutions.
U.S. Pat. No. 5,510,245 (1996; Magers) and U.S. Pat. No. 5,326,697 (1994; Magers) disclose an improved calorimetric method that utilizes a reductive pathway based on lipoamide dehydrogenase (LADH) and a thiol-sensitive indicator dye such as Ellman's reagent. It was found the NADH, produced from the β-hydroxybutyrate dehydrogenase enzyme reaction, can interact with lipoamide dehydrogenase (LADH) and D,L-lipoamide to form a thiol compound (6,8-dimercaptooctamide). The 6,8-dimercaptooctamide then interacts with a thiol-responsive indicator dye such as Ellman's reagent. Upon reaction, the thiol-sensitive indicator dye undergoes a detectable color transition that can be used to measure the level of 3-hydrobutyrate in the blood sample.
The colorimetric methods for 3-hydrobutyrate suffer the disadvantages of poor stability, interference from co-existing species such as ascorbate, glutathione etc. in the blood, and insufficient sensitivity and accuracy.
NAD- and NADP-dependent enzymes are of great interest insofar as many have substrates of clinical value, such as glucose, D-3-hydroxybutyrate, lactate, ethanol, and cholesterol. Amperometric electrodes for detection of these substrates and other analytes can be designed by incorporating this class of enzymes and establishing electrical communication with the electrode via the mediated oxidation of the reduced cofactors NADH and NADPH.
NAD- and NADP-dependent enzymes are generally intracellular oxidoreductases. The oxidoreductases are further classified according to the identity of the donor group of a substrate upon which they act. The category of oxidoreductases is also broken down according to the type of acceptor utilized by the enzyme. The enzymes of relevance have NAD+ or NADP+ as acceptors. These enzymes generally possess sulphydryl groups within their active sites and hence can be irreversibly inhibited by thiol-reactive reagents such as iodoacetate. An irreversible inhibitor forms a stable compound, often through the formation of a covalent bond with a particular amino acid residue that is essential for enzymatic activity.
U.S. Pat. No. 6,541,216 (2003; Wilsey et al.) discloses a biosensor and method to test blood ketone bodies using an amperometric meter. The test strip has a reagent that is reactive with β-hydroxybutyrate in sample solution to generate an electrical output signal, which is related to the concentration of β-hydroxybutyrate in the sample solution. The reagent in this method includes ferricyanide salt as mediator, β-hydroxybutyrate dehydrogenase as the first enzyme operative to catalyze the oxidation of β-hydroxybutyrate, NAD+ as a cofactor corresponding to the first enzyme, and diaphorase as the second enzyme operative to catalyze the oxidation of a reduction form of the cofactor (NADH). The oxidation form of the mediator will accept the electron from the second enzyme and generates an electrical signal at the electrode surface, which is related to the concentration level of β-hydroxybutyrate.
U.S. Pat. No. 6,736,957 (2004; Forrow et al.) and a research paper (N.J. Forrow et.al, Biosensors & Bioelectronics, 2005, 20, 1617-1625) disclose an amperometric biosensor for β-hydroxybutyrate based on the discovery of NAD+ and NADP− mediator compounds that do not bind irreversibly to thiol groups in the active sites of intracellular dehydrogenase enzymes. These mediator compounds such as 1,10-phenanthroline quinone (1,10-PQ), which is used as an electron mediator in their electrochemical measurement system, can increase the stability and reliability response in amperometric electrodes constructed from NAD- and NADP-dependent enzyme. The dry reagents include 1,10-phenanthroline quinone (1,10-PQ), β-hydroxybutyrate dehydrogenase and NAD+ as the cofactor. This sensor shows reliable and sensitive response to the concentration levels of β-hydroxybutyrate in blood samples. Meldola's Blue (MB) was also studied as a mediator in the system, but it was found that MB did not work well in their electrochemical test system due to the inhibition of β-hydroxybutyrate dehydrogenase enzyme activity by MB and poor long term stability of the test strips.
The dehydrogenase enzymes such as, for example, glucose dehydrogenase, D-3-hydroxybutyrate dehydrogenase (HBDH), and lactate dehydrogenase et.al are known to be common dehydrogenases for construction of biosensors. As disclosed by Forrow et al., there are certain mediators that are considered efficient mediators for NADH but are irreversible enzyme inhibitors such as Meldola's blue, 4-methyl-1,2-benzoquinone (4-MBQ), 1-methoxy phenazine methosulphate (1-Meo-PMS) and 2,6-dichloroindophenol (DCIP), which cause losing the activity of enzymes, insensitive response and poor stability in sensors containing dehydrogenase enzymes.